The invention relates to a method and device for the measurement of chemical and/or biological samples, in particular with the use of luminescence spectroscopy.
A known method for the measurement of such samples is the screening of the samples. In screening, the interaction between two bio-molecules in the presence of a test substance is studied. The bio-molecules can be, for example, the pairs of ligand-receptor, substrate-enzyme, protein-protein, or protein-DNA. A measurable signal may be produced either by the bio-molecules themselves, or, as in most cases, sample markers have to be bound to the bio-molecules. As markers, substances are employed that produce signals based on radio activity, luminescence or absorption. When color markers are used that produce luminescence, the color markers are excited by electromagnetic radiation, e.g. suitable laser light, so that the electromagnetic radiation lifts an electron to a higher energy level and the color marker gives off light, i.e. luminesces, by the electron returning to its original energy level. The probability of the return of the electron to its original energy level, and thus of the emission of luminescence, is exponentially distributed overtime. The mean duration of the exited state is therefore also referred to as the luminescence life. Since, in most cases, luminescent markers have but a slight influence on the interactions between bio-molecules and are extremely sensitive compared to other markers, the use of luminescent color markers is particularly advantageous. Information on reactions between two bio-molecules are obtained by establishing a relation between the change in the light emitted by the color markers and the reaction of the bio-molecules.
In an example of a measuring method, a target molecule is first exposed to a fluorescent reagent as a luminescent reagent adapted to bind to the target molecule. If, for example, the intensity of the fluorescence changes during the binding, it can be used to quantify the binding. In another experiment, the target is exposed to both the fluorescence-marked reagent and a single substance. If a binding between the substance and the target occurs, the fluorescence-marked reagent is separated from the target. Thus, the ratio of bound and free marked molecules changes. In turn, this entails a change in the emission of fluorescence by the sample and it can be assessed whether and in how far a substance binds to the target.
In order to increase the amount of information to be acquired during a measurement, a plurality of markers, in particular two color markers, may be used. Depending on the excitation energy of the two color markers, two electromagnetic radiation sources, e.g. lasers, of different wavelengths are employed to excite the color markers. For example, a red and a green color marker is used in combination with a red and a green laser. When red and green color markers were used, it has been found that, when a red laser was used together with a green laser, the intensity of the light emitted by the red color marker is less than the intensity of the red color markers, when the same is irradiated exclusively with red laser light. This loss of intensity entails a loss of information and leads to falsified results.
This phenomenon can also be found when color markers of different colors are used.